Sutterella species have been frequently associated with human diseases, such as autism, Down syndrome, and inflammatory bowel disease (IBD), but the impact of these bacteria on health still remains unclear. Especially the interactions of Sutterella spp. with the host are largely unknown, despite of the species being highly prevalent. In this study, we addressed the interaction of three known species of Sutterella with the intestinal epithelium and examined their adhesion properties, the effect on intestinal barrier function and the pro-inflammatory capacity in vitro. We also studied the relative abundance and prevalence of the genus Sutterella and Sutterella wadsworthensis in intestinal biopsies of healthy individuals and patients with celiac disease (CeD) or IBD. Our results show that Sutterella spp. are abundant in the duodenum of healthy adults with a decreasing gradient toward the colon. No difference was detected in the prevalence of Sutterella between the pediatric IBD or CeD patients and the healthy controls. Sutterella parvirubra adhered better than the two other Sutterella spp. to differentiated Caco-2 cells and was capable of decreasing the adherence of S. wadsworthensis, which preferably bound to mucus and human extracellular matrix proteins. Furthermore, only S. wadsworthensis induced an interleukin-8 production in enterocytes, which could be due to different lipopolysaccharide structures between the species. However, its pro-inflammatory activity was modest as compared to non-pathogenic Escherichia coli. Sutterella spp. had no effect on the enterocyte monolayer integrity in vitro. Our findings indicate that the members of genus Sutterella are widely prevalent commensals with mild pro-inflammatory capacity in the human gastrointestinal tract and do not contribute significantly to the disrupted epithelial homeostasis associated with microbiota dysbiosis and increase of Proteobacteria. The ability of Sutterella spp. to adhere to intestinal epithelial cells indicate that they may have an immunomodulatory role. Sutterella, consisting of Sutterella wadsworthensis, Sutterella parvirubra, and Sutterella stercoricanis, have been isolated from the human gastrointestinal (GI) tract and canine feces  and healthy children  is generally characterized by a decrease in the abundance of Firmicutes and an increase of Proteobacteria, the phylum that also contains lla spp. . In a hella spp. . Increaslla spp. . TherefoSutterella spp. in the human GI tract still remain unclear. In this study, we addressed the adhesive and pro-inflammatory properties of three known Sutterella spp. using various in vitro models, including the adhesion to intestinal epithelial cell lines, mucus and extracellular matrix (ECM) proteins, as well as their effect on the epithelial barrier function and interleukin-8 (IL-8) release in enterocytes. In addition, we investigated the relative abundance of these species in different parts of the human intestine and the prevalence in pediatric patients with CeD or IBD.The epithelial interactions and the role of Sutterella wadsworthensis , S. parvirubra  and S. stercoricanis  were obtained from German Collection of Microorganisms and Cell Cultures (DSMZ) and grown on Gifu anaerobic medium  agar for 2 days at 37°C in an anaerobic incubator  containing 85% N2, 10% CO2, and 5% H2. Bacteroides fragilis E-022248T received from the VTT Culture Collection  was grown under anaerobic conditions for 2 days at 37°C on Brucella agar with hemin and vitamin K  along with 5% defibrinated sheep blood . Escherichia coli K12-derived TOP10 cells  were aerobically cultivated in Luria–Bertani broth  overnight at 37°C with agitation (220 rpm). For adhesion experiments, Sutterella spp. were grown on GAM agar supplemented with 10 μl ml-1 of [6′-3H]thymidine  to metabolically radiolabel the cells.2. Cells were passaged after reaching 80% confluence using HyClone HyQTase  to detach the cells. Passage numbers 5–28 were used in the experiments. Caco-2 cells were grown in RPMI 1640 medium  supplemented with heat-inactivated (30 min at 56°C) fetal bovine serum , non-essential amino acids , 15 mM HEPES , 100 U ml-1 penicillin and streptomycin , and 2 mM L-glutamine . HT-29 cells were cultivated in McCoy 5A  medium containing 10% FBS and 100 U ml-1 PEST. For adhesion and ELISA assays, 10,000 Caco-2 or HT-29 cells per well were seeded onto 96-well microplates.The human colonic epithelial cell lines Caco-2 (ACC 169) and HT-29 (ACC 299) were obtained from DSMZ and grown at 37°C in an incubator under oxic atmosphere with 5% COHuman colonic mucus was isolated from a healthy piece of tissue from a colorectal cancer patient as previously described . The useSutterella spp. and B. fragilis bacterial masses were collected from agar plates with an inoculation loop and suspended to lysis buffer. E. coli broth culture was centrifuged  to pellet the cells and DNA was extracted with QIAamp DNA mini kit  according to the manufacturer’s instructions. DNA concentrations were measured using NanoDrop ND-1000 spectrophotometer .Clostridium difficile infection , who underwent diagnostic nasogastroduodenoscopy or/and colonoscopy and were found to have a healthy intestine (Supplementary Table n = 33) and patients with CeD (n = 9), Crohn’s disease , and ulcerative colitis . The age and gender of the subjects, the location of biopsies, and disease activity are compiled in Supplementary Table Intestinal mucosal biopsies from pediatric patients were obtained from our previous studies , 2014 orSutterella spp. and S. wadsworthensis from the intestinal biopsy samples was carried out using previously described quantitative real-time PCR (qPCR) assays (Sutterella genus assay was prepared from amplified 260-bp region between V6 and V8 of the 16S rRNA gene from S. wadsworthensis (ATCC 51579). Primers were SuttFor (5′-CGC GAA AAA CCT TAC CTA GCC-3′) and SuttRev (5′-GAC GTG TGA GGC CCT AGC C-3′)  and reverse primer SWR (5′-CTG GCA TGT CAA GGC TAG GT-3′)  (6–101 copies). Samples were analyzed in triplicate using a 96-well plate format. Amplification and detection of DNA by qPCR were performed with Mx3005P Real-time PCR System  and the results were analyzed with MxPro Real-time PCR Software version 4.1 . AmpliTaq Gold DNA Polymerase kit  and SYBR Green chemistry were used. Each 25 μl reaction contained 25 ng of biopsy DNA, 0.5 μM of each primers, 0.1 mM deoxynucleoside triphosphate (dNTP) mix , 10 mM Tris–HCl (pH 8.3), 50 mM KCl, 3 mM MgCl2, 1:75,000 dilution of SYBR Green I , and 0.025 U μl-1 AmpliTaq Gold DNA Polymerase. The thermocycling parameters consisted of an initial denaturation step at 95°C for 10 min, followed by 45 cycles of 95°C (30 s), 58°C (1 min), and 72°C (1 min).Quantification of ) assays . The staGC C-3′) . For S. G GT-3′) . The staC AG-3′)  and 338RC AG-3′) . All theSutterella spp. to Caco-2 and HT-29 cell lines , mucus, and ECM proteins was studied as described previously . Porcine mucus (Sigma-Aldrich) and human ECM proteins, including collagens I  and IV (Sigma-Aldrich), laminin (Sigma-Aldrich), and fibronectin , were diluted in PBS. Bovine serum albumin  and fetuin (Sigma-Aldrich) were used as controls for non-specific binding. Human and porcine mucus (50 μg well-1 in PBS) and ECM proteins (2.5 pmol well-1 in PBS) were incubated on Maxisorp microtiter plates overnight at 4°C and blocked with 0.5% (w/v) BSA in PBS for 1 h prior to the adhesion assay. Four technical replicates  were used in each experiment. [3H]Thymidine-labeled S. wadsworthensis, S. parvirubra, and S. stercoricanis cells were collected from agar plates to PBS, washed with an appropriate medium (McCoy 5A for HT-29 cells and RPMI 1640 for Caco-2) without supplements or PBS and adjusted to OD600nm 0.25, which corresponded to approximately 108 cells ml-1. The bacterial suspensions were incubated for 1 h on mucus and ECM proteins at 37°C or on the cell lines at 37°C in a CO2 incubator, followed by washing three times to remove the non-adherent cells. Bound bacteria were lysed with 1% SDS–0.1 M NaOH and radioactivity was measured with a liquid scintillator . The percentage of adhered bacteria was calculated relative to the radioactivity of the bacterial suspension initially added to the wells. The adhesion to ECM proteins was also calculated as percentages and compared to the level of background binding (BSA).The adherence of eviously . BrieflyS. parvirubra and radiolabeled S. wadsworthensis were performed as previously described with minor modifications  were used in each experiment. Eight days post-plating Caco-2 and HT-29 cells  were fixed with 4% paraformaldehyde  for 2 h at room temperature before the assay. The cell monolayers were fixed due to multiple, repeated washing steps, which otherwise cause detaching of the non-fixed cells. In the competition assays, both species were incubated with the enterocytes together, whereas in the exclusion assays S. parvirubra was incubated first, then washed away and followed by S. wadsworthensis. In the displacement assays S. wadsworthensis adhered first followed by S. parvirubra. The percentage of adhered bacteria was calculated relative to the radioactivity of the bacterial suspension initially added to the wells and the non-competitive situation with similar washing steps is shown as the control.The displacement, exclusion, and competition assays with ications . The bacSutterella spp. on transepithelial electrical resistance (TER) of Caco-2 monolayer was determined as previously described  was calculated by multiplying the tissue resistance values by surface area of the filter membrane.The impact of escribed . The Cacescribed . E. colintegrity . B. frag control . The blaSutterella spp., E. coli and B. fragilis was carried out as previously described  were used in each experiment. OptEIA Human IL-8 ELISA kit  was used according to the manufacturer’s instructions to measure the concentration of the chemokine in the culture media. To prepare the LPS preparations, bacteria were adjusted to OD600nm 0.25 and 500 μl of the suspension was centrifuged  to pellet the cells. The pellets were suspended to 20 μl of lysis buffer  and heated to 100°C for 10 min. After cooling, proteinase K  was added to the cell lysates with a final concentration of 0.5 mg ml-1 and incubated at 60°C for 2 h. The lysates were once again heated to 100°C to inactive proteinase K, then 450 μl of McCoy 5A medium supplemented with 10% heat-inactivated FBS was added and the lysates were diluted to 1:10–1:10,000.IL-8 assays were only performed using the HT-29 cell line, because Caco-2 cells are known to be unresponsive to LPS stimulation possibly due to defects in TLR4 signaling . The meaescribed . In brieSutterella spp. and E. coli TOP10 were visualized with silver staining as previously described (-1) at 60°C for 2 h, followed by heat-inactivation of the enzyme. The samples were run in SDS-PAGE under denaturing conditions on a gradient gel  and silver stained. Proteinase K treatment reduces bacterial proteins to small peptides but leaves the carbohydrate LPS unaffected, which then can be detected due to periodate-sensitive cis-glycols in the core and the side-chains of LPS . Briefly, the bacterial pellets were run in SDS-PAGE as explained above without the proteinase K treatment and blotted onto polyvinylidene difluoride Immobilon P membranes . After the E. coli LPS antiserum, the membrane was incubated with 1:100,000 diluted HRP-conjugated goat anti-rabbit IgG secondary antibody . Amersham ECL Advance Western blotting detection kit  was used according to the manufacturer’s instructions.The LPS structures of escribed . Prior ts of LPS . The LPSt-test was used to determine significant differences as compared to the control. Homoscedasticity testing was performed with Levene’s test to identify equal or unequal variances. A paired-sample t-test was used for pre- and post-FMT samples. Statistical differences between the samples were tested with one-way analysis of variance using a Bonferroni post hoc. All statistical analyses were carried out with IBM SPSS Statistics program version 21.0 . A p-value of <0.05 was considered statistically significant.All the experiments were done using three to four technical replicates depending on the assay and repeated two to three times  to confirm the results. Different cultures of bacteria and different passages of cell lines were used in the separate experiments. The following tests were used to compare samples within an experiment. A two-sample Sutterella spp. in the different parts of a healthy intestine, mucosal biopsies from duodenum, ileum, and rectum were analyzed with Sutterella-specific and universal qPCR assays. The results were calculated as a relative proportion of Sutterella spp. compared to all bacteria . Adult patients who underwent successfully the FMT therapy for a rCDI were also included in the study and rectal samples both pre- and post-FMT were analyzed. We observed a decreasing gradient of Sutterella spp. along the GI tract from the small intestine to the large bowel. The relative abundance of Sutterella in the duodenum and ileum was significantly higher (p = 0.005) as compared to the rectum, while the percentage of these species of the whole duodenal microbiota seemed to vary greatly between individuals. The pre- and post-FMT samples of rCDI patients (p = 0.443) or the samples from rCDI patients and other study subjects did not differ in their Sutterella abundance . A gradient in the relative abundance of Sutterella spp. along the GI tract was observed also at individual level in the case of one subject, from whom biopsies of all three intestinal locations were obtained. In these samples Sutterella spp. constituted 1.3, 0.3, and 0.2% of the total microbiota in the duodenum, ileum, and rectum, respectively.In order to assess the amount of Sutterella spp. and specifically S. wadsworthensis are differently present in the duodenum or colon of pediatric patients with CeD or IBD compared to the healthy controls. The 16S rRNA gene copy numbers in these samples were too low for reliable quantitation and, therefore, only qualitative analysis could be performed. The PCR analysis showed that Sutterella spp. were present in half of healthy children’s duodenum and in six out of nine CeD patients . Surprisingly, S. wadsworthensis was not detected in the duodenal biopsies of either group. The biopsies from pediatric UC or CD patients and the controls were obtained from different parts of the colon , whereas the prevalence of S. wadsworthensis was 29–40% (p = 0.799). Furthermore, there was no statistical difference in the prevalence of Sutterella spp. or S. wadsworthensis when comparing the UC and CD patients and the healthy controls or depending on the intestinal biopsy location.Next, we studied whether S. wadsworthensis, S. parvirubra, and S. stercoricanis to immobilized human and porcine mucus, as well as to ECM proteins, including type I and IV collagens, laminin, and fibronectin. The adherence ability of S. wadsworthensis to porcine mucus was better compared to the other two species . In fact, S. parvirubra did not bind mucus, since less than 1% of the added cells had adhered which is considered to be non-specific background binding. The same differences between S. wadsworthensis and the other species were also observed in the adhesion to human mucus . Sutterella spp. showed no difference between human and porcine mucus binding. When studying the adherence of these bacteria to ECM proteins, fetuin as a highly glycosylated protein and BSA as a non-glycosylated protein were included in the assay as controls. S. wadsworthensis showed significantly higher adhesion to laminin, collagen I, and fibronectin compared to the background-level . Moreover, its adherence to BSA was 2.3%, whereas the same adhesion ratios for S. parvirubra and S. stercoricanis were merely 0.8 and 1.5%, respectively (data not shown). S. parvirubra could only bind to laminin and collagen I, and S. stercoricanis to collagens I and IV .We examined the adherence of Sutterella spp. to IECs, we used the Caco-2 and HT-29 cell lines as models of enterocytes. Caco-2 cells start to differentiate 5–7 days post-plating and both cell lines lack the mucus layer covering the intestine. Cells at three different growth stages were included in the assays: undifferentiated (3 days post-plating) as well as 8- and 21 -day-old cells . Interestingly, S. parvirubra adhered significantly better to 8- and 21-day-old Caco-2 cells compared to the other two species. There was no difference in the adhesion of all three species to HT-29 cells except S. parvirubra adhered better than S. stercoricanis to 3-day-old cells. In the displacement, exclusion, and competition assays, the effect of S. parvirubra on the adherence of S. wadsworthensis was tested using 8-day-old cell lines. The adhesion was significantly lowered (p < 0.001) to 55 and 52% in competition to Caco-2 and HT-29 cells, respectively (Table 2). Furthermore, displacement by S. parvirubra reduced the binding of S. wadsworthensis to both enterocyte lines (p < 0.05) but the effect was more profound in Caco-2 cells (73%). The preliminary results on the capability of S. parvirubra to exclude S. wadsworthensis showed merely a 15% reduction in the adherence to Caco-2 cells and no effect on the adherence to HT-29 cells (data not shown).To compare the adhesion capability of ld cells . HT-29 cSutterella spp. on the epithelial barrier function, we measured TER of a Caco-2 monolayer co-cultured with the bacteria. TER of 8-day-old Caco-2 cells (5 days after confluence) was measured at 24 h intervals for 48 h in total. Partially differentiated Caco-2 monolayer was used to imitate the intestinal conditions comprising of enterocytes still in the process of maturation and prone to changes of barrier integrity due to external stimuli . On the contrary, already within 24 h B. fragilis (positive control) had greatly enhanced the monolayer integrity, whereas E. coli (negative control) had significantly decreased it.In order to study the impact of  stimuli . SuttereSutterella spp. by incubating 10-fold dilutions of the bacteria with HT-29 monolayer followed by measurement of IL-8 release from the cells. Again, E. coli and B. fragilis were used as controls, whereas the cells incubated solely with the culture medium functioned as a background control. For E. coli, the results of 1:10 dilution are not shown due to excess stimulation. Previously B. fragilis has been shown not to induce an IL-8-response in HT-29 cells . E. coli and S. wadsworthensis, on the other hand, induced a significant, dose-dependent IL-8 response in the HT-29 cells, although for S. wadsworthensis the effect was only reached with 1:10 and 1:100 dilutions.We examined the potential pro-inflammatory properties of 29 cells . SimilarSutterella spp. and E. coli. All three Sutterella spp. induced an IL-8 production above the medium control level with 1:1,000 dilution but not with 1:10,000 dilution . The effect was stronger with the LPS preparation from S. wadsworthensis. The same LPS preparations were run in SDS-PAGE gel and visualized with silver staining to compare the LPS structures  database and BLAST  for genes that encode enzymes for Kdo2-lipid A modification. We found out that Sutterella spp. lack the genes for LpxL and LpxM acyltransferases, which are present in E. coli (Table 3).The same assay was carried out with crude LPS preparations of Sutterella spp. is substantial in the duodenum of adults with a decreasing gradient toward the colon, which has not been demonstrated before. The bile resistant and microaerophilic properties of Sutterella spp.  and after a successful FMT treatment of rCDI. Before FMT, rCDI patients have a dysbiotic microbiota with a higher abundance of Proteobacteria and facultative anaerobes, which are decreased to normal level by FMT together with an increase of obligately anaerobic gut commensals , core-oligosaccharide and O-antigen repeats and recognized by the host TLR4–MD2 complex  and observed that these species lack the genes for LpxL and LpxM, which are the enzymes responsible for adding the secondary acyl chains (hexa-acylation), and, consequently, are likely to carry the tetra-acylated lipid A with reduced toxicity. Our results support this view, since Sutterella LPS did not produce a strong IL-8 response in the HT-29 cells in contrast to E. coli. Quite surprisingly only E. coli and related Proteobacteria have all nine genes needed for the complete Kdo2-lipid A biosynthesis (Bacteroides dorei, which has tetra- and penta-acylated lipid A, differs majorly in its immunomodulation capacity from that of E. coli LPS (Sutterella spp.We next searched the genomes of ynthesis . Recentlcoli LPS . The strin vitro Sutterella spp. can be successfully grown only on solid media or in chopped meat broth, which includes solid particles (Sutterella spp. possess specific niches in the small intestine. S. parvirubra adhered strongest to enterocytes and showed competitiveness against S. wadsworthensis in enterocyte binding, whereas S. wadsworthensis displayed the strongest binding to intestinal mucus and ECM proteins. The human GI tract can be colonized by single or multiple Sutterella spp. (S. parvirubra with low pro-inflammatory capacity is tissue-associated and S. wadsworthensis with the ability of inducing inflammation cascades occupies the mucus layer. Proteobacteria most likely are involved in the pro-inflammatory changes leading to dysbiosis in the human GI tract (Sutterella spp. do not seem to contribute to a dysbiotic state of the gut, but instead their presence, especially in the duodenum, and ability to adhere to IECs indicate them to be mutualistic keeping the immune system alerted.It is of interest that articles . This malla spp. . ConsideGI tract . Yet, itGI tract . HoweverSutterella are widely prevalent commensals with IEC-adhesion and mild pro-inflammatory capacities in the human GI tract.In conclusion, our results indicate that the members of the genus KH, RS, and VK conceived the study. KH and VK performed the experiments. KH and RS interpreted the data and wrote the manuscript. MK and PA recruited patients as well as collected clinical samples and data. All authors critically revised the manuscript for intellectual content and approved the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The Multidimensional Daily Diary of Fatigue-Fibromyalgia-17 instrument (MDF-Fibro-17) has been developed for use in fibromyalgia (FM) clinical studies and includes 5 domains: Global Fatigue Experience, Cognitive Fatigue, Physical Fatigue, Motivation, and Impact on Function. Psychometric properties of the MDF-Fibro-17 needed to demonstrate the appropriateness of using this instrument in clinical studies are presented.N = 381). Confirmatory factor analyses (CFA) were performed to ensure understanding of the multidimensional domain structure, and a secondary factor analysis of the domains examined the appropriateness of calculating a total score in addition to domain scores. Longitudinal psychometric analyses  were also conducted on the data from Baseline to Week 6.Psychometric analyses were conducted to evaluate the factor structure, reliability, validity, and responsiveness of the MDF-Fibro-17 using data from a Phase 2 clinical study of FM patients  and non-normed fit index (NNFI) were 0.997 and 0.992 respectively, standardized root mean square residual (SRMR) was 0.010 and the root mean square error of approximation (RMSEA) was 0.06. In addition, total score (CFI and NNFI both 0.95) met required standards. For the total and 5 domain scores, reliability and validity data were acceptable: test-retest and internal consistency were above 0.9; correlations were as expected with the Global Fatigue Index (GFI) (0.62-0.75), Fibromyalgia Impact Questionnaire (FIQ) Total (0.59–0.71), and 36-Item Short Form Health Survey (SF-36) vitality (VT) (0.43–0.53); and discrimination was shown using quintile scores for the GFI, FIQ Total, and Pain Numeric Rating Scale (NRS) quartiles. In addition, sensitivity to change was demonstrated with an overall mean responder score of -2.59 using anchor-based methods.The MDF-Fibro-17 reliably measures 5 domains of FM-related fatigue and psychometric evaluation confirms that this measure meets or exceeds each of the predefined acceptable thresholds for evidence of reliability, validity, and responsiveness to changes in clinical status. This suggests that the MDF-Fibro-17 is an appropriate and responsive measure of FM-related fatigue in clinical studies. Fibromyalgia (FM) is a disorder characterized by chronic widespread pain and tenderness that is estimated to affect 0.5–10% of the worldwide population, with approximately 2–3%  of the affected individuals present in the United States (US) alone , 7. BeyoThere is a growing body of evidence from both clinical and regulatory communities supporting FM-related fatigue as a multidimensional concept , 9–11. AExisting research that had been conducted with FM patients for concept elicitation , cognitiThe original qualitative work confirmed the content validity of the instrument,  developeThe original pool of 23 items developed from the qualitative work was incorporated into a Phase 2 study of TD-9855, an investigational norepinephrine and serotonin reuptake inhibitor, in patients with FM . PatientPatients were instructed to complete all items at approximately the same time every evening, and a restricted time-window for completion was programmed between the hours of 17:00 and 24:00. Retrospective completion of missed days was not allowed. The diary questions were presented sequentially and the option to skip items was not provided.Each item was presented as a 0–10 NRS anchored by “not at all” at 0 and “extremely” at 10; higher scores indicated greater fatigue severity for 22 of the 23 items. A weekly score was calculated as the mean of the available data if greater than 4 entries were completed within the 7-day period. Observations less than 4 entries were considered missing with no imputation. All items were evaluated on an item level to confirm the hypothesized 5 domain, and a 17-item fit of the data to the conceptual model identified previously in qualitative work . The 5 dA number of additional instruments were included in the study and used to inform the psychometric evaluation of the MDF-Fibro-17  to assess the degree to which the hypothetical conceptual measurement model fit the data.The factor structure of the MDF-Fibro-17 items was evaluated using the 17-item, five-factor latent-model . The averaged domain raw scores were used as the manifest variables in a single-factor CFA.Following the initial CFA conducted to explore the multidimensional domain structure of the measure, a secondary factor analysis of the domains was conducted to explore the appropriateness of calculating a total score Fig. . This seFor the initial and secondary CFA, the goodness of fit of the models was evaluated by several fit indices using the following pre-defined thresholds: a comparative fit index (CFI) of 0.95 or higher; a root mean square error of approximation value (RMSEA) of 0.06 or lower; a non-normed fit index (NNFI) of 0.90 or higher; and a standardized root mean residual (SRMR) of 0.08 or lower –23. ConfThe relationships between individual items and the proposed MDF-Fibro-17 domains were evaluated. Item-total correlations, within the hypothesized domains, were expected to be 0.4 or greater –26.The consistency of the items to measure fatigue at individual time points as well as the repeatability while patients were considered stable were evaluated. Reliability of the MDF-Fibro-17 domain and total scores were assessed using test-retest reliability  and internal consistency  , 24. TheConvergent validity was assessed by looking at correlations with other measures of fatigue . A moderate relationship (>0.4) was expected with overall FM severity , and measures of physical functioning on the SF-36 physical functioning (PF) subscale and physical component score (PCS).Divergent validity was assessed by looking at correlations with measures assessing concepts other than fatigue, such as mood (HADS), sexual function (ASEX), and cognitive function  and other aspects of HRQoL measured on the remaining 6 subscales on the SF-36v2.Moderate or greater correlations (>0.4) were expected to confirm convergent validity, and weaker correlations (<0.4) expected to confirm divergent validity. However, given the complex relationships between symptoms in FM, correlations with measures assessing concepts other than fatigue were not expected to be zero. These analyses were conducted on absolute scores at Baseline and repeated at End of Study using change scores calculated for each measure.Known-groups validity was examined to provide further evidence of construct validity. Scores on measures indicative of overall severity of condition , and the GFI, a measure of fatigue, were divided into quintiles. Mean MDF-Fibro-17 total and domain scores were computed for each quintile. A generalized linear model provided an overall F-test for the group discrimination with effect size estimates considered as 0.2 , 0.5 (moderate), and 0.8 (large) .Effect sizes are defined as the mean change found in a variable divided by the standard deviation (SD) of that variable. Effect sizes are used to translate “the before and after changes” into a standard unit of measurement that will provide a clearer understanding the relative sensitivity and performance of each clinical variable. The ability of the MDF-Fibro-17 to detect changes observed in the clinical study was evaluated using distribution- and anchor-based methods. Distribution-based methods include estimations based on observed variance in the sample such as the evaluation of ½ SD or 1 standard error of measurement. Anchor-based methods allow for the conceptual linking  between additional known clinical or patient variables.For the distribution-based analyses, 2 definitions for the ½ SD approach were used: ½ of the baseline SD and ½ of the change score SD; and 2 for the standard error of the mean (SEM) approach: SEM based on the ICC (test-retest coefficient and the baseline SD), and SEM based on the ICC and the change score SD –30.For the anchor-based analyses, a collapsed PGI-C scale category of “very much improved” and “much improved” versus remaining PGI-C responses denoting minimal improvement, no change, or decline  was used for discrimination on the MDF-Fibro-17  software Version 9.1.3 . Values reported in text are means ± SD.The final sample of 392 patients in the intention-to-treat (ITT) population  had an average age of 45.7 ± 10.6 years. The majority of patients were Caucasian (82.7%) followed by Black/African American (13.0%)  patients from the ITT population had data available on the DFS-Fibro at Baseline. This analysis set was used in the psychometric evaluation of the measure.The items were administered via electronic PDA, which did not allow items to be skipped; therefore, there no missing data were at the item level. No floor or ceiling effects at the item level were observed (0.3-1.3% and 0.3–0.5% respectively). All items showed a negative skew, with the majority of values to the right of the mean. Nine items had a z-score greater than 2.0 indicating a substantial departure from normality.N = 192) concluded that the MDF-Fibro-17 fit the data well with all parameters met the pre-specified criteria. The initial CFA model was evaluated on the 17 items, 5-factor model hypothesized for the MDF-Fibro 17 and suggest that the model fit the data from both studies. These results are presented in Table An initial CFA conducted using preliminary baseline data from the TD-9855 Phase 2 study (N = 381), the averaged domain raw scores were used as the manifest variables in a single-factor CFA to explore the appropriateness of a total score. The CFA models were evaluated in a stepwise fashion to allow for accumulation of evidence surrounding the dimensionality of the MDF-Fibro-17. The single-factor CFA model was evaluated on 5 domain scores and the total of 5 domain scores and suggest that the model fit the data. The CFI and NNFI were both 0.952, above their respective 0.95 and 0.90 required thresholds. The SRMR was 0.020, below the prespecified 0.08 threshold, which is, in part, due to the small number of parameters in this model. Due to the presence of correlated residuals between Fatigue Experience and the Physical Fatigue domain items, the RMSEA (0.15) was short of recommended standards and was associated with a notably high modification index . The path coefficients for the 5 domain MDF-Fibro-17, before accounting for correlated residuals, were 0.92 for Global Fatigue Experience, 0.88 for Cognitive Fatigue, 0.87 for Physical Fatigue, 0.98 for Motivation and 0.99 for Impact on Function.Using data collected in the full-dataset TD-9855 Phase 2 study  was well under the required threshold, and the RMSEA (0.061) also met required standards. The path coefficients for the 17-item, 5 domain MDF-Fibro, accounting for correlated residual between Global Fatigue Experience and Physical Fatigue 0.42), were between 0.88 and 0.990. The correlation coefficients for individual items ranged from 0.92 to 0.99. The CFA results are shown in Table 2, were bCorrected item-total correlations within hypothesized domains ranged from 0.92 to 0.96 for Global Fatigue Experience, 0.96 to 0.98 for Cognitive Fatigue, 0.85 to 0.91 for Physical Fatigue, 0.94 to 0.96 for Motivation, and 0.93 to 0.97 for Impact on Function, all of which met pre-defined criteria and were considered substantial. For all items except two, observed correlations were highest with its own domain compared to with other domains. Item “How tired did your body feel today?”, part of the Physical Fatigue domain, correlated more strongly with Global Fatigue Experience (0.92), Motivation (0.88), and Impact on Function (0.88) than its own domain (0.85). Item “How much did tiredness make it difficult to do things today?”, part of the Impact on Function domain had a slightly higher correlation with the Motivation domain than its own domain (0.95 versus 0.93). All correlations are presented in Table Test-retest reliability was assessed by evaluating the reproducibility of MDF-Fibro-17 scores over the time period between Baseline and Day 8 and from Week 5 to Week 6. All ICCs (Spearman Brown) exceeded the required 0.70 level, for baseline versus day 8, ICCs ranged from 0.71 to 0.82 (median of 0.74), and for Week 5 versus Week 6 all exceeded 0.90.Internal consistency was confirmed as acceptable with strong Cronbach’s alpha for the total score and all domain scores (ɑ = 0.94-0.99). Reliability data are shown per MDF-Fibro domain in Table These data indicate overall good construct validity for the MDF-Fibro-17. Correlations with measures hypothesized to capture the same or a highly related concept, demonstrating convergent validity, were moderate (>0.4) to high (>0.7) at Baseline and End of Study for MDF-Fibro-17 scores. The highest correlations for each of the MDF-Fibro-17 total and domain scores were with the GFI 0.62 to 0.84), the FIQ Total (0.59 to 0.81), and the SF-36 VT (0.43 to 0.68). The majority of the correlations with the SF-36 measures of physical functioning – the PF and PCS – were all at least moderate with the exception of the MDF-Fibro-17 Cognitive Fatigue domain at Baseline versus PF and PCS (-0.31 and -0.28 respectively), and the MDF-Fibro-17 Global Fatigue Experience, Cognitive Fatigue, Physical Fatigue, and Motivation domains against the SF-36 PF at End of Study . The results for convergent validity are presented in Table 2 to 0.84With respect to divergent validity, weaker correlations were observed, with low correlations (<0.4) between all MDF-Fibro-17 total and domain scores versus sexual function (ASEX) at Baseline and End of Study, and all measures of cognitive function , mood (HADS), and the other SF-36 subscales at Baseline. Low to moderate correlations were observed at the End of Study Treatment visit . The results for divergent validity are presented in Table p < 0.001) when performed using quintiles. Large effect sizes (>0.8),[All known-group difference analyses of MDF-Fibro-17scores were highly significant (s (>0.8),, 41 detep < 0.001) changes were observed in all MDF-Fibro-17 scores from Baseline to End of Study. A medium effect size (>0.5) was observed for the Cognitive Fatigue domain (-0.69). Effect sizes for the total score and all other domains were large (-0.85 to -0.95). Similar effect sizes to those observed on the MDF-Fibro-17 were also observed in the pain intensity NRS, FIQ total score, GFI, and SF-36 VT.Significant , GFI (>11-point improvement), and FIQ total score (>8-point improvement) were similar to those determined by selected distribution based methods (-2.55 to -2.94). However, the responder definitions determined using the PGIC much improved category, GFI, and FIQ had a broader range (-2.06 to -3.41). The mean responder score, based on the anchor-based analyses, for the MDF-Fibro-17 Total Score and the 5 domains ranged from -2.48 to -2.85. Overall, the recommended responder cut-off for the total score as well as the other domains is -2.5 . The analyses confirmed the domain structure suggested by the conceptual model developed from in-depth qualitative work with FM patients, and indicated sound psychometric properties of the measure.All 17 items in the MDF-Fibro-17 performed well as individual items and as part of the 5 domain structure of the instrument. The multidimensional structure allows the MDF-Fibro-17 to capture the broad experience of FM-related fatigue, a characteristic that has been identified as important within the clinical and regulatory community , 10. In Tests of internal consistency and test-retest reliability were strong, indicating that this is a highly reliable measure. The correlations observed between the MDF-Fibro-17 and other measures in the study hypothesized to be either similar  or dissimilar  were overall as expected, confirming good construct validity. The strongest relationships were observed between the MDF-Fibro-17 and overall FM severity  and the GFI, another measure of fatigue. The moderate correlations with the SF-36 VT, a single item evaluating a simple concept similar to fatigue, and some of the measures for divergent validity demonstrate the high level of complexity of FM-related fatigue, in which multiple symptoms are experienced and, though distinct, are closely related.Known-groups analysis revealed that the MDF-Fibro-17 total and domain scores were able to differentiate between all groups tested. Highly significant changes were observed over the study period on all scores of the MDF-Fibro-17, with medium to large effect sizes, which reflected the changes observed on other outcomes in the study, indicate that the instrument is sensitive to detecting changes observed in a clinical study.Responder analyses conducted using different definitions for both anchor based and distribution-based techniques produced similar estimates and the results suggested a reasonable responder cut-off to be around -2.5.One limitation to this study is that although the MDF-Fibro-17 has the potential to assess the different components of FM-related fatigue based on data described above, this study was conducted in a particular clinical trial population in response to drug therapy intervention. Therefore, responsiveness and sensitivity to other therapies would need to be further explored in future studies.The psychometric evaluation and strong evidence of content validity indicate that the MDF-Fibro-17 is a relevant, psychometrically robust, multidimensional instrument, with sensitivity to detection change and clear response definitions. Taken as a whole, the MDF-Fibro-17 has the potential to become a reliable clinical outcome assessment tool to evaluate fatigue in adult patients with FM within a clinical trial setting .


CA 19-9 is used as a tumor marker in colon, pancreas, biliary, and gastric cancers. Laryngeal cancer is the most common malignant epithelial tumor among head and neck cancers and has no specific tumor marker.A 66-year-old male patient had severe reflux symptoms during 5 years and had an isolated CA 19-9 elevation. Follow-up analysis revealed that he had larynx cancer and after laryngectomy, CA 19-9 levels decreased to normal range.Currently, CA 19-9 is not a marker for malignancy. Laryngeal carcinoma has no specific tumor marker, but laryngeal squamous cell carcinoma may be manifested by elevated CA 19-9 levels.How to cite this article: Özkan H, Karakaya F, Karaeren Z, Perçinel S. Persistent Elevation of CA 19-9 Levels in the Long-term Follow-up before Laryngeal Cancer. Euroasian J Hepato-Gastroenterol 2017;7(1):92-94. It is usually used for the diagnosis of pancreatobiliary and gastrointestinal cancers, and especially as a combination with radiological methods to be helpful for follow-up after treatment.23 Laryngeal cancer is the most common malignant epithelial tumor among head and neck cancers. The most important risk factor is smoking and alcohol consumption, and the risk improves substantially when both factors are combined. The condition is more common in patients above 50 years of age and it is also more frequently seen in males. Over 95% of laryngeal cancers are squamous cell carcinomas. Histologically, they can rarely be adenocarcinomas, sarcomas, and neu-roendocrine tumors. There are no specific tumor markers for the prognosis and follow-up of laryngeal cancer.4CA 19-9, which is used as a tumor marker, was first developed in the 1980s as a monoclonal antibody against Sialyl-Lewis A antigen. It is known to be produced in normal cells  and also to have an increased production when sialyl transferase enzyme is inhibited.A 66-year-old male patient was admitted to our institution after he underwent follow-up in another institution. CA 19-9 levels were found to be elevated. He had no known diseases other than hypertension. There were no alarming symptoms in his systemic examination, such as weight loss, abdominal pain, hematemesis, and night sweats. He had no history of alcohol consumption and abstained from smoking for last 20 years, after smoking for 20 years. The patient was found to have chronic reflux symptoms, which did not regress with proton pump inhibitors. The patient had undergone a gastrectomy and a billroth 1 anastomosis long time ago. No pathological findings were observed in the initial laboratory tests; both biochemical parameters and total blood counts were normal, including hemoglobin, which was found to be 16.6 gm/dL. The tumor marker tests were renewed and CA 19-9 was 193 U/mL, carcinoembryonic antigen was 2 ng/mL, alpha-fetoprotein was 4 ng/mL, and CA 72-4 was 1.1 U/mL, which showed that the patient had an isolated CA 19-9 elevation. To rule out a possible malignity concerning the gastrointestinal system, a gastrointestinal system endoscopy and colonoscopy were performed. Endoscopic and colonoscopic examinations had no findings pointing to a malignity. A former gastric operation scar was observed at the upper gastrointestinal endoscopy and the lower esophageal sphincter was found to be relaxed, explaining the reflux symptoms. To rule out a possible pancreatobiliary system malignity, abdominal computerized tomography and magnetic resonance cholangiopancreatography were performed, but no findings related to a malignity that can be at the pancreas or the biliary system were observed in the radiological imaging. As the patient had persistent CA 19-9 elevation with no pathological findings to explain this situation, a follow-up was initiated. As the patient had severe reflux symptoms, during the next 5 years, three gastrointestinal endoscopies were performed. In the endoscopic examination, similar findings were observed with the prior endoscopy. Again, during the follow-up, as the CA 19-9 levels were not stable, another colonoscopic examination was performed, but no findings pointing to a malignity were observed. During this period, the patient was examined with an abdominal ultrasound once in every year but no malignity-related findings were observed. During these years, although CA 19-9 was always 5 to 15 times the normal value, no other tumor markers were found to be elevated . Due to 3 CA 19-9, being produced as a monoclonal antibody against Sialyl-Lewis A antigen, apart from the known cancer localizations, is shown to do more color uptake in the squamous cell laryngeal carcinoma rather than normal larynx tissue (5 In our case, the persistent CA 19-9 is thought to be the cause of the laryngeal mucosa being edematous as a result of severe reflux. Also, focal CA 19-9 expression detected immunohistochemically by the tumor cells and serum CA 19-9 level lowering to normal values show that the source of the tumor marker is the laryngeal tissue. In conclusion, even though CA 19-9 is not a specific tumor marker, i.e., put forward for laryngeal cancers, persistent CA 19-9 elevation should remind the clinician for laryngeal cancer during differential diagnosis, after gastrointestinal and pancreatobiliary cancers are ruled out (Generally, tumor markers are used to screen a possible disease relapse for patients with malignancy diagnosis. Tumor markers are not distinctly specific to a type of cancer and they are also produced in cells that belong to healthy individuals. As the tumor biology differs in every patient, tumor marker elevation does not result in the same way for each patient. The tumor markers may even be normal, even though the same cancer types are observed in the patients.x tissue . CA 19-9uled out .We assume that a laryngeal squamous cell carcinoma may produce CA 19-9. Currently, CA 19-9 is not a specific marker for a malignancy so that it cannot be used for diagnosis of any tumor. But it should be kept in mind that laryngeal squamous cell carcinoma can be a reason of elevated CA 19-9 levels except gastrointestinal system tumors.


Pseudomonas aeruginosa PAO1 against the antimicrobial agent were monitored by confocal laser scanning microscope (CLSM). The microfluidic sensor along with 1-TDPPOin this study exhibits the following advantages: (i) Real-time chemo-repellent responses of cell dynamics; (ii) Rapid eradication of biofilm by embedded obstacles and powerful antibacterial agents, which significantly reduce the response time compared to classical methods; (iii) Minimal consumption of cells and antimicrobial agents; and (iv) Simplifying the process of the normalization of the fluorescence intensity and monitoring of biofilm by captured images and datasets.This paper describes the use of an analytical microfluidic sensor for accelerating chemo-repellent response and strong anti-bacterial 1-(Thien-2-yl)-3- prop-2-en-1-one (1-TDPPO). The chemically-synthesized antimicrobial agent, which included prop-2-en-1-one and difluoro phenyl groups, was moving through an optically transparent polydimethylsiloxane (PDMS) microfluidic sensor with circular obstacles arranged evenly. The response, growth and distribution of fluorescent labeling  Pseudomonas aeruginosa PAO1 is a threat to human health because it tends to form agglomerates that encase themselves in the extracellular matrix of various polysaccharides and proteins [Microorganisms have the ability to detect or respond to specific chemical gradients . Bacteriproteins . As a reproteins ,7,8. ForSeveral methods have been developed for the analysis of chemical interaction with bacteria, including agarose, collagen, stopped-flow diffusion chamber, and capillary assays ,10,11,12Pseudomonas aeruginosa PAO1 and an antimicrobial agent was observed directly by a fluorescence intensity surface plot, providing information on the bacterial distribution from achieved images inside the microfluidic reactor. A stable biofilm growth and a quick response of the antimicrobial agent were induced by artificially-embedded obstacles using the volume analysis method. The growth and eradication of biofilm was a major determinant for the antimicrobial efficacy, whereas the efficacy of microfluidic sensor was determined by the response time.The purpose of this study is to focus on the fact that the newly invented 1-TDPPO is superior to the existing chemical agent , and this effect is in turn enhanced by the microstructures embedded in the microfluidic system. 1-TDPPO, a newly synthesized antimicrobial agent, was used and based on the fact that prop-2-en-1-one or difluoro phenyl exhibited a very strong antimicrobial efficacy ,38,39. A1H NMR : 6.96 , 7.18 , 7.32 , 7.71 , 7.85 , 7.91 ; 13C NMR : 111.9, 127.2, 128.3, 129.9, 131.2, 132.2, 134.3, 145.4, 161.0, 163.0, 182.2. All of the compounds were added directly to the growth medium at the time of inoculation. As shown in Pseudomonas aeruginosa PAO1, which encased themselves in the extracellular matrix of various polysaccharides and proteins. This result indicated that it took three days to confirm its strong antimicrobial property, despite its outstanding antimicrobial properties. In the case of the Petri dish system, molecular diffusion, the dominant mixing process, proceeds relatively slow and inhibits efficient mixing. This simply demonstrates the superiority of our microfluidic system, which induced a fast response compared to a three-day reaction time. The mixing performance by embedded circular microstructures was compared by microchannel without microstructures in 1-TDPPO was examined by adding the synthesized compound by inoculation to reach final concentrations of 0, 2.5, 5, 10, 20, and 30 µM to determine the ranges of concentrations. P. aeruginosa PAO1 passing through a microfluidic chip, and the actual observed the pixel intensity (R.F.U.) was 233 without 1-TDPPO. Strong fluorescence intensity was observed when P. aeruginosa PAO1 moved throughout the microfluidic sensor. This means that the microfluidic sensor can be used to observe the real-time response of bacteria against the antimicrobial agent. In contrast to P. aeruginosa PAO1 activity performed at three different positions , as shown in P. aeruginosa PAO1 bacteria were located in the inlet, whereas there was a small number of bacteria in the outlet due to the antimicrobial agent. Throughout this process, the passive chemo-repellent gradient could be achieved within a very short period of time using a simple method because the embedded circular obstacles in the microfluidic reactor led to enhanced mixing efficiency without requiring a gradient generator. This device for real-time monitoring of cell dynamics can also be reused after washing.After determining the concentration of and injecting the 1-TDPPO, the real-time cell dynamics were observed. P. aeruginosa PAO1 biofilm appeared to be relatively constant from 24 h until day 5, as the three-dimensional profiles of the fluorescence intensity did not provide a characteristic difference. The images shown in P. aeruginosa PAO1 biofilm existing on the microfluidic chip based upon position. The difference in the biofilm thickness was less than 5% in all cases. From the given data, an analysis of the biofilm in microfluidic sensors along with CLSM can be considered a reliable method and is expected to increase the use of this system.Bacteria aggregates began to be detected at 12 h and biofilm growth became significant within 24 h, as shown in Pseudomonas aeruginosa PAO1 was investigated and compared with those of 20% and 90% ethanol solutions, as shown in Pseudomonas aeruginosa PAO1 and totally removed them. In addition, the increasing concentration of antimicrobial agent  resulted in a better performance. Tiny residuals of Pseudomonas aeruginosa PAO1 were present in the microfluidic sensor when it was operated for 5 min with 1-TDPPO, as shown in Pseudomonas aeruginosa PAO1 finally disappeared when the mixing time was increased to 7 min, as shown in The real-time cell monitoring of 1-TDPPO provided a clue that it can be useful in eradicating the biofilm. The efficacy of 1-TDPPO for the eradication of P. aeruginosa PAO1 wild-type strain. The bacteria were transformed by electroporation with a plasmid, which stably expressed a green fluorescent protein (GFP). P. aeruginosa PAO1 was grown in a Luria–Bertani (LB) medium supplemented with carbenicillin (50 μg/mL) for recombinant selection and retention. The stationary phases used for column chromatography  and thin-layer chromatography (TLC) plates (Silica-gel 60 F254) were purchased from Merck KGaA. All other chemicals were obtained from Sigma-Aldrich . The final concentration of bacteria in this study was 1 × 109 cells/mL.The bacterial strain used in this study was the 1H nuclear magnetic resonance (NMR) and 13C NMR spectra were recorded on a JNM-ECA 500 spectrophotometer (Jeol) at 500 and 125 MHz, respectively. The internal standard used for these spectra was tetramethylsilane (TMS). The IR spectra (KBr) were recorded on a Bruker-Vector 22 instrument. The molecular mass of the compounds was derived from the high-resolution mass spectral (HRMS) technique using a JEOL JMS600 instrument.Aqueous sodium hydroxide (0.011 Mol) at 0 °C was added to a mixture of 2-acetylthiophene  and 2,6-difluorobenzaldehyde  in ethanol. The solution was stirred for 6–8 h at room temperature. Upon completion of the reaction according to TLC, the reaction mixture was poured into ice-cold water, and the pH of the mixture was then adjusted to 6 by adding 0.01 N HCl solutions. The obtained precipitates were filtered, dried and re-crystallized from the mixture to obtain the compound. The melting points were determined in open capillary tubes and were uncorrected. The P. aeruginosa PAO1 was seeded onto a solid agar plate using the spread plate method. 10 μL (106 cell/mL) of P. aeruginosa PAO1 was cultured at 37 °C for 24 h to induce cell growth in the solid medium. The compounds were placed on a tested bacteria-seed plate. The anti-bacterial assay plate was again incubated at 37 °C for 24 h. The solvent without the compounds was used as a control sample to compare the antimicrobial efficacy.The anti-bacterial activity was pre-examined using the halo method on a solid agar plate. A microfluidic chip with/without circular obstacles in the micro-channels was fabricated using standard photoresist-based soft lithography . A positThe analyses of both the antimicrobial activity and population of bacteria were measured by Nikon Eclipse Ti-E spinning disk confocal microscopy . Fluorescence was stimulated at a 488 nm wavelength, and a filter isolated the emitted wavelength between 505 and 539 nm, which is the green portion of the visible spectrum . A serieTwo Tygon tubings  were pre-cleaned with ethanol and distilled water before being connected by a syringe pump  to the microfluidic chip at the two microfluidic chip inlets, as shown in P. aeruginosa was introduced into inlet A and the synthetic antimicrobial agent was injected into inlet B. All experiments were conducted at room temperature. To observe a real-time dynamic response of cells against the antimicrobial agent, the employed flow rate was 0.5 μL/min using a syringe pump (±0.5% accuracy) to observe a chemo-repellent gradient within the microfluidic sensor. The optimal selection of the flow rate is the limiting point of this technique. We performed the chemical gradient on injecting 1-TDPPO at flow rates of 0.05 µL/min to 5.0 µL/min and selected the optimal flow rate of 0.5 µL/min. The criteria used were a compromise between high flow rates that occasionally interrupt the bacterial motility, and low flow rates that do not obtain a linear chemical gradient. As a result, preliminary tests were performed on the microfluidic channels, and the optimal flow rate, 0.5 μL/min, was selected. The bacterial responses and their distribution profiles through the microfluidic sensor were observed directly by CLSM. A three-dimensional image of the fluorescence intensity profile was proportional to the metabolically-active population of bacteria which applied the principle as two-dimensional bacterial responses [Pseudomonas aeruginosa PAO1-GFP in the microfluidic system was incubated at 37 °C for 7 days before the determination of colony-forming unit (CFU) per mL. Luria-Bertani (LB) media and Pseudomonas aeruginosa PAO1 were initially introduced to the two inlets until the microfluidic reactor was filled. The formation of the Pseudomonas aeruginosa PAO1 biofilm was monitored and analyzed continuously by CLSM using the volume analysis method. After biofilm formation, deionized water was injected into the microfluidic channels to remove the dead bacteria. 1-TDPPO was then introduced into inlets at 0.5 μL/min. The change of biofilm was also measured by CLSM. To compare the efficacy of 1-TDPPO, popular antimicrobial agents, 20% and 90% ethanol, were used in this study. It was known that a low concentration of 20% ethanol demonstrated that all cells embedded in the biofilm were killed [−4~10−3), in which the antimicrobial agent was transported to the surface by diffusion. The prepared esponses . Throughe killed . Accordie killed ,50. Anote killed ,51. UndeP. aeruginosa PAO1 using a new synthetic chemical, 1-TDPPO, which has organic compounds of prop-2-en-1-one and difluoro phenyl. The combination of two organic compounds exhibits a stronger antimicrobial efficacy than the more commonly used antibacterial agents. The bacterial distribution in response to antimicrobial agents was observed directly by CLSM using the intensity surface plot, which provided a high-throughput chemo-repellent gradient. Microfluidic sensors for generating gradients offer spatial and temporal profiles in a predictable and controllable manner and can be compensated using conventional methods. A rapid response of cell dynamics in microfluidic sensors, which provides information about cell response, was directly observed. It eventually saved a lot of processing time for the normalizing of fluorescence intensity, which was used by the software program. It was clear that P. aeruginosa PAO1 exhibited a strong tendency to repel 1-TDPPO in the channel inlet and was depleted completely in the channel outlet. P. aeruginosa PAO1 was formed in clusters within 24 h and exhibited a significant change in fluorescence intensity in 6 days, as observed using microfluidic sensors. The growth of the biofilm was observed by CLSM using a three-dimensional volume analysis method, which replaced the numerical models for measuring the depth of the biofilm. The microstructures embedded in the flow-based microfluidic sensor showed superior antibacterial activity compared to no microstructures. Using a microfluidic sensor embedded in microstructures and synthetic 1-TDPPO, the biofilm was eradicated in a short time and exhibited outstanding antimicrobial efficacy. 20% and 90% ethanol solutions led to successful performances of eradicating biofilm formation, but sufficient mixing time should be considered in advance.This study proposed and examined the potential use and merits of microfluidic sensors for the direct analysis of cell response, biofilm formation and eradication of


Mycoplasma hyopneumoniae is a colonizing respiratory pathogen that can cause great economic losses to the pig industry worldwide. Although putative virulence factors have been reported, the pathogenesis of this species remains unclear. Here, we used the virulent M. hyopneumoniae strain 168 to infect swine tracheal epithelial cells (STEC) to identify the infection-associated factors by two-dimensional electrophoresis (2-DE). Whole proteins of M. hyopneumoniae were obtained and compared with samples cultured in broth. Six differentially expressed proteins with an increase in abundance of ≥1.5 in the cell infection group were successfully identified. A String network of virulence-associated proteins showed that all the six differential abundance proteins were involved in virulence of M. hyopneumoniae. One of the most important upregulated hubs in this network, elongation factor thermo unstable (EF-Tu), which showed a relatively higher expression in M. hyopneumoniae-infected STEC and obtained a higher score on mass spectrometry was successfully recombined. In addition to its canonical enzymatic activities in protein synthesis, EF-Tu was also reported to be located on the cell surface as an important adhesin in many other pathogens. The cell surface location of EF-Tu was then observed in M. hyopneumoniae with flow cytometry. Recombinant EF-Tu (rEF-Tu) was found to be able to adhere to STEC and anti-rEF-Tu antibody enclosed M. hyopneumoniae decreased adherence to STEC. In addition, surface plasmon resonance (SPR) analysis showed that rEF-Tu could bind to fibronectin with a specific and moderately strong interaction, a dissociation constant (KD) of 605 nM. Furthermore, the block of fibronectin in STEC also decreased the binding of M. hyopneumoniae to the cell surface. Collectively, these data imply EF-Tu as an important adhesin of M. hyopneumoniae and fibronectin as an indispensable receptor on STEC. The binding between EF-Tu with fibronectin contributes to the adhesion of M. hyopneumoniae to STEC.HIGHLIGHTSM. hyopneumoniae.Elongation factor thermo unstable (EF-Tu) exists on the cell surface of M. hyopneumoniae.EF-Tu moonlights as an adhesin of The adhesive effect of EF-Tu is partly meditated by fibronectin. Mycoplasma hyopneumoniae is the primary pathogen responsible for swine enzootic pneumonia. This infection is highly prevalent (ranging between 38 and 100%) in almost all areas of pig production worldwide, and causes significant economic losses , which is one of the target tissues of M. hyopneumoniae. Importantly, the results of this study could facilitate uncovering new biological activities or unknown functions of known proteins that can lead to a more complete understanding of the virulence-associated proteins and the complex biological and infectious processes taking place during infection. For example, the canonical function of the cytoplasmic protein, EF-Tu is acting as a GTPase on the binding of aminoacyl-tRNA to the ribosome-mRNA complex during translation elongation in protein synthesis . All experimental procedures conformed to the guidelines of Jiangsu Province Animal Regulations (Government Decree No. 45) in accordance with international law.M. hyopneumoniae strain 168 (GenBank accession CP002274) was originally isolated in 1974 from an Er-hua-lian pig  exhibiting typical clinical and pathogenic characteristics of swine mycoplasmal pneumonia in Gansu Province, China  containing 20% (v/v) swine serum at 37°C  and inoculated with M. hyopneumoniae strain 168. A series of 1:10 dilutions of cultures in broth with a metabolic indicator was used to estimate titers of M. hyopneumoniae strain 168. The dilution of the last tube to show growth was taken as the number of CCU (color change unit)  = 20] were washed with sterile PBS and resuspended in RPMI-1640 medium with 2% (v/v) FBS and incubated with STEC for 48 h. Supernatants were collected from each well for M. hyopneumoniae separation. M. hyopneumoniae strain 168 was cultured in RPMI-1640 medium with 2% (v/v) FBS in 24-well plates for use as the control. The assay was performed three times.STEC were seeded in 24-well plates and cultured in RPMI-1640 medium  supplemented with 10% (v/v) fetal bovine serum . The cells were grown at 37°C in 5% COTM Protein Assay Kit .The obtained culture was centrifuged at 12,000 rpm for 20 min at 4°C. The precipitates were washed three times with sterile 10 mM Tris-HCl (pH 7.4) and resuspended in 30 μL protein extract . After vortexing for 15 s, the mixture was placed in an ice bath for 30 s, and the procedure was repeated for 15 min. The whole protein in the supernatant was obtained by centrifugation at 12,000 rpm for 30 min at 4°C. A ReadyPrep 2-D cleanup kit  was used to clean up the protein samples. The protein concentration was determined using a BCAThe purified proteins were quantified and redissolved with 350 μL rehydration solution , and then centrifuged  to remove insoluble components. The samples were loaded into a 17-cm strip , and positively rehydrated at 50 V for 12 h at 20°C. Isoelectric focusing (IEF) was then carried out at 20°C as follows: slow to 250 V for 1 h; rapid to 1,000 V for 1 h; linear to 10,000 V for 3 h; rapid to 10,000 V to total 90,000 Vh; rapid to 500 V for the preservation of samples. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed according to a previously described protocol  analysis . Peptide mass fingerprinting data were analyzed using the MASCOT server (http://www.matrixscience.com). Peptides with a rank of 1 in the MASCOT search were considered significant and used for the combined peptide score.After Coomassie blue staining, the protein spots from each gel were detected and matched automatically by the PDQuest V8.0 software followed by additional visual analysis. The intensity of individual spot for each gel was normalized relative to total valid spot intensity to eliminate gel-to-gel variation. Only spots whose abundance changed by ≥1.5-fold and a value of M. hyopneumoniae and to filter the most important virulence associated factors, we summarized the putative virulence factors reported in the previously published papers , defining a “confidence score” that can be used to evaluate the confidence in the interaction. The interactions derived from textmining and experiments with a confidence score of at least 0.4 were considered for analysis. The protein-protein interaction network was visualized using Cytoscape (3.5.1).To determine how the differential proteins contribute to virulence in ef-tu gene encoding one of the most important novle virulence associated factors was cloned into pET-32a using homologous recombination technology . The sequence that overlapped with the end of the cloning site was added onto the insert through a PCR step. Table Escherichia coli BL21 (DE3) for isopropyl-β-d-thiogalactopyranoside (IPTG)-inducible expression, and the induced proteins were purified by Ni-chelating chromatography . Polyclonal antibody against the recombinant EF-Tu (rEF-Tu) was prepared by subcutaneously immunizing 1-month-old New Zealand white rabbits. Each rabbit was immunized a total of three times with 1 mg of purified recombinant protein emulsified in Freund's adjuvant  at 2-week intervals. Sera were collected 1 week after the third immunization. The animals used in this study met the legal and ethical requirements and were treated humanely.The M. hyopneumoniae incubated with STEC is expressed as the percentage of that in M. hyopneumoniae cultured in KM2 medium and the three replicates were subjected to statistical analysis by SPSS 20.0.Equal amounts (40 μg for each lane) of the protein samples were separated on a 12% SDS-PAGE gel. The proteins were electrophoretically transferred onto polyvinylidene fluoride (PVDF) membranes  and developed with Ponceau-S as the loading control. After washing with TBST buffer  and blocking with TBST buffer containing 5% skimmed milk, the membranes were incubated with primary antibody against rEF-Tu , followed by horseradish peroxidase (HRP)-conjugated secondary antibody . Signals were detected with the ECL substrate  using the ChemiDoc XRS+ system . Image J software was used to calculate the optical density (OD) of the corresponding bands. The OD of EF-Tu from different samples was normalized to that of the Ponceau-S stained membrane. The level of abundance of EF-Tu in M. hyopneumoniae strains. In brief, three pathogenic M. hyopneumoniae strains, 168, NJ and WX (108 CCU) were incubated with anti-rEF-Tu primary serum at a 1:100 dilution (preimmune rabbit serum was used as the negative control). The blank control was incubated with PBS instead of antibody. Then, M. hyopneumoniae cells were stained with fluorescein isothiocyanate (FITC)-conjugated anti-IgG. Finally, the fluorescence intensity was detected using a flow cytometer (BD Accuri® C6)  were washed three times with PBS and pre-incubated with the polyclonal antibody (1:20 dilution) against rEF-Tu at 37°C for 30 min. M. hyopneumoniae that had been pre-incubated with preimmune sera (1:20 dilution) were used as the control. The bacterial suspension in RPMI-1640 medium was added to each well of 24-well cell plates with confluent STEC, and the plates were centrifuged at 800 × g for 10 min and incubated at 4°C for 2 h. Following incubation, the wells were washed three times with PBS to remove unbound M. hyopneumoniae cells. Then the cells in the wells were treated with lysis buffer containing 0.1% trypsin and 0.025% (v/v) Triton X-100, followed by bacterial genome extraction and real-time PCR for bacteria counting. Real-time PCR was performed using a QuantStudio® 5 Real-Time PCR System according to a previous method, with a slight modification  was performed. Total 20 μg recombinant proteins, including rEF-Tu and a negative control MRP-D2, which was previously verified not to bind to fibronectin  experiment on a Biacore™ X100 Plus instrument . Fibronectin was diluted to 10 μg/mL in 10 mM sodium acetate (pH 4.0) and covalently linked to the carboxylmethylated dextran matrix of a sensor chip CM5 as ligands using an amine coupling kit (Biacore AB). Immobilization of soluble fibronectin generated resonance units (RU) of 2868. The binding kinetics was measured with the increasing concentrations (0–200 μg/mL) of the analyte (rEF-Tu) in running buffer  surfactant P20]; Biacore AB) at a flow rate of 30 μL/min for 180 s over immobilized fibronectin at 20°C. The dissociation phase was monitored for 1,000 s by allowing buffer to flow over the chip. Association kinetics were analyzed manually using Biacore™ X100 Control Software  were washed three times with PBS. The bacteria resuspended in RPMI-1640 medium was added to the 24-well cell plates and incubated at 4°C for 2 h. After incubation, the wells were washed three times with PBS to remove unbound M. hyopneumoniae cells. Then the cells in the wells were treated with lysis buffer containing 0.1% trypsin and 0.025% (v/v) Triton X-100. The following bacterial genome extraction and real-time PCR for bacteria counting were conducted according to the method in section Adherence Inhibition Assay of Antibody Against rEF-Tu. The assay was performed in triplicate, and the data were analyzed using Student's t-test using SPSS 20.0. A value of p ≤ 0.05 was considered statistically significant.The confluent STEC in 24-well cell plates was washed three times with PBS and pre-incubated with the antibody against fibronectin (1:20 dilution) at 37°C for 30 min. The RPMI-1640 medium was used instead of fibronectin antibody as the negative control. M. hyopneumoniae-infected STEC were subjected to MALDI-TOF-MS/MALDI-TOF-TOF-MS analysis. Six novel differential abundance proteins were successfully identified. They were YX2, pyruvate dehydrogenase E1-alpha subunit, EF-Tu, hypothesis protein MHJ-0662, enolase, and adenine phosphoribosyltransferase. Among them, EF-Tu showed a relatively higher expression in M. hyopneumoniae-infected STEC in 2D gels  in the M. hyopneumoniae virulence and to filter the most important virulence associated factors. The results revealed a total of 32 direct physical interactions among the 14 nodes  by which the EF-Tu surface protein affected virulence, we used indirect immunofluorescence to determine whether or not rEF-Tu could adhere to STEC. Significant fluorescence was detected on the cell surface of STEC incubated with rEF-Tu Figure ; meanwhiM. hyopneumoniae. Polyclonal antibody against rEF-Tu and preimmune sera were used to incubate with M. hyopneumoniae cells respectively before adherence to STEC. The level of adherence of M. hyopneumoniae incubated with anti-rEF-Tu sera is expressed as the percentage of M. hyopneumoniae adherence with preimmune sera. The results revealed that after incubation with anti-rEF-Tu antibody, the adherence efficiency of M. hyopneumoniae to STEC showed a 46% (P < 0.05) reduction compared to that incubated with preimmune sera  and to fibronectin, while no specific reaction was observed in the negative control MRP-D2, a non-related recombinant protein which was verified not to bind to fibronectin in a previous study  reduction compared to that incubated without antibody . These results demonstrate that M. hyopneumoniae may increase abundance of EF-Tu to help better attach to the host cells to some extent. Thus, M. hyopneumoniae EF-Tu, in addition to its major cytoplasmic, biosynthetic, and metabolic roles, can translocate to the surface and moonlight as an important adhesion factor.EF-Tu is the most abundant bacterial protein . Interaction with the ECM components, with the most common being fibronectin, may influence the initiation, facilitate colonization, and establishment of infection, and express important virulence factors (Patti and Höök, M. hyopneumoniae adherence to cell surface significantly. Immunohistochemical staining of bronchioles showed that fibronectin is more abundant in the ciliary borders of the bronchioles, which is a prefered place for M. hyopneumoniae colonization. Thus, fibronectin was verified one of the host cell receptors for M. hyopneumoniae adhesion.Using far-WB analysis, rEF-Tu was found to be able to bind to fibronectin. The dissociation constant of rEF-Tu determined by SPR displayed values with a specific and moderately strong interaction. Furthermore, block of fibronectin in STEC decreased M. hyopneumoniae. Further analysis revealed that M. hyopneumoniae displays EF-Tu on the surface to bind to host fibronectin with a moderately high affinity, thereby promoting adherence to STEC. This binding was markedly reduced by pretreatment of bacterial cells with anti-rEF-Tu sera or pretreatment of STEC with anti-fibronectin antibody. The binding between EF-Tu with fibronectin was proven to contribute to the adhesion of M. hyopneumoniae to STEC.In summary, using system-wide methodologies, EF-Tu was found to act as an important hub in the virulence-associated network of M. hyopneumoniae, it can be speculated that when EF-Tu is blocked, the adhesion of M. hyopneumoniae still partly exists.However, in the competitive adhesion inhibition assay, only partial inhibition of EF-Tu was observed. Since several other proteins, such as P97 (Zhang et al., M. hyopneumoniae virulence. A lot remains to be understood about how proteins, especially those lacking signal motifs, localize on bacterial cell surfaces. M. hyopneumoniae moonlighting proteins and their roles in infection and immunity should be further studied in the future.In conclusion, moonlighting proteins located in various parts of the cell, not only in the cytoplasm but also on the cell membrane, have been suggested to be associated with YY completes the study of the pathogenic mechanism of EF-Tu and prepares the manuscript. The comparative proteomics analysis is done by HW. JW, and BL perform the cell adhesion experiments. MW prepares the polyclonal antibody. ZF, JX, and GS modify the manuscript. QX and ML supervise and guide this work.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Evaluate the predictive value of the preoperative blood neutrophil-to-lymphocyte ratio (NLR) on the clinical outcomes of patients with gastric neuroendocrine neoplasms (g-NENs) after radical surgery.P < 0.05). A higher blood NLR was not significantly associated with clinical characteristics . According to the multivariate analysis, the NLR was an independent prognostic factor of RFS and OS. Nomograms, including the NLR, Ki-67 index and lymph node ratio, had superior discriminative abilities to predict clinical outcomes. The recurrence rate was 37% (55/147). The median time to recurrence was 9 months; 48 (87%) patients experienced recurrence within the first 2 years. Both the NLR and Ki-67 index were correlated with liver metastases (both P < 0.05) and were also negatively correlated with recurrence time (both P < 0.05).The NLR was significantly higher in patients with g-NENs than in matched normal volunteers (We enrolled 147 patients who were diagnosed with g-NENs and underwent radical surgery. Receiver operating characteristic curve analysis was used to identify the optimal value for blood NLR. Univariate and multivariate survival analysis were used to identify prognostic factors for g-NENs. A nomogram was adopted to predict RFS and OS after surgery.As an independent prognostic factor for g-NENs, blood NLR can improve the predictability of RFS and OS. We recommend that g-NEN patients with a high blood NLR or high Ki-67 index undergo surveillance during the first month and then every 3 months for 2 years post-surgery. Gastric neuroendocrine neoplasms (g-NENs) are a type of relatively rare tumors mainly derived from enterochromaffin-like cells (ECL-cells) localized in the gastric mucosa . AlthougP > 0.05, Figure P < 0.001, Figure P < 0.001, Figure In total, 147 normal volunteers (NVs) with similar age and gender proportions as the neoplasm patients were enrolled (both P < 0.05). However, the multivariate analysis revealed no significant differences in the above clinicopathological factors between the two groups .The univariate analysis revealed Table  that a hAs shown in Figure P < 0.05, Table P < 0.05, Table P < 0.05, Table To investigate which parameters were associated with clinical outcomes, a univariate survival analysis and a multivariate survival analysis for RFS and OS were performed. The univariate analysis identified larger tumor size, presence of postoperative complications, greater invasion depth, higher LNR, higher Ki-67 index, and higher blood NLR as prognostic indicators for poorer RFS . Both the blood NLR and Ki-67 index were negatively correlated with the recurrence time . The median time to recurrence was 9 (range 1–56) months, and 87% (48/55) of patients experienced recurrence within the first 2 years, and only 7% (4/55) of patients experienced recurrence > 3 years after surgery Figure . As showP < 0.001). Similar results for recurrence rate were also observed between the high Ki-67 index group and the low and intermediate Ki-67 index groups (P = 0.023). Additionally, elevated blood NLR was significantly associated with both liver metastasis and peritoneal metastasis (both P < 0.05), whereas only liver metastasis was significantly correlated with a high Ki-67 index (P = 0.008).Details regarding the recurrence site following surgery were listed in Table Neuroendocrine neoplasms, particularly g-NENs in the digestive system, are a unique subgroup of tumors with great clinical heterogeneity and varied biology. Recently, the incidence of g-NENs, which account for 6% of neuroendocrine neoplasms in all systems, has gradually increased due to improvements in diagnostic techniques and increasing knowledge regarding g-NENs . In a liIn recent years, pathological staging and the Ki-67 index were viewed as valuable prognostic factors for patients with g-NENs. Deep tumor invasion, the presence of lymph node metastasis and distant metastasis, and a high Ki-67 index were associated with decreased long-term survival . In thisPostoperative local recurrence and distant metastasis are the leading causes of death for malignant tumor patients. In this study, the 5 years recurrence rate for patients with g-NENs after radical surgery was 37%. Liver metastasis was the most common type of recurrence, whereas the spleen, kidney, and brain were relatively rare sites of recurrence. Our results were similar to previous reports [As most of the current guidelines recommend, postoperative surveillance for g-NENs begin at 3 to 12 months after radical surgery and the intervals vary from 6 to 12 months , 5, 28. This study had some limitations. This study was uncontrolled and performed within a single institution. The results should be confirmed by subsequent prospective studies. There were some heterogeneity in this study, such as histological type . Through accumulating more cases, we will focus on just one of these three types in the future. However, to our knowledge, our study enrolled more patients with g-NENs than similar literature reports and demonstrated that the blood NLR was able to predict long-term survival relatively accurately for patients for the first time. Our study could be the basis for a subsequent prospective clinical study.In conclusion, as a simple, easily measurable, and inexpensive inflammatory biomarker, a preoperative blood NLR is an independent predictor of RFS and OS, and its combination with the Ki-67 index and LNR could improve prognosis prediction in g-NENs patients undergoing radical surgery. For patients with a blood NLR > 2.20 or a Ki-67 index > 20%, we recommend a modified surveillance strategy starting at the first month post-surgery and at intervals of 3 months for the first 2 years after surgical resection.n = 11), perioperative death (n = 1), and incomplete/inaccurate medical records (n = 14). A total of 147 patients who underwent curative-intent surgery for g-NENs were enrolled in this study. The pathological data for these patients were reconfirmed by two pathologists according to the North American Neuroendocrine Tumor Society (NANETS) guidelines (2010) [A total of 173 patients diagnosed with g-NENs at Fujian Medical University Union Hospital between March 2006 and March 2015 were identified from a prospective database. The exclusion criteria for this study were as follows  . In totaThe preoperative blood cell counts that were collected closest to the date of surgery were obtained from the electronic patient record system in our hospital. The blood NLR was calculated as the absolute neutrophil count divided by the absolute lymphocyte count. A Receiver operating characteristic (ROC) curve analysis was performed in relation to the presence of recurrence and death from any cause. For all 147 patients, a blood NLR of 2.20 had the highest sensitivity and specificity for both outcomes. Therefore, patients were categorized into two groups: low blood NLR (≤ 2.20) with 77 patients and high blood NLR (> 2.20) with 70 patients.2O2 in methanol. The nonspecific binding was blocked by incubation using 1% bovine serum albumin  in phosphate buffered saline (PBS). Subsequently, the slides were incubated overnight at 4°C with primary monoclonal mouse antibody against Ki-67 . Normal goat serum was used as a negative control. After washing with PBS, tissue sections were incubated with secondary antibody for 20 minutes at room temperature and then stained with diaminobenzidine (DAB). Finally, the slides were counterstained with hematoxylin, dehydrated, and mounted with a coverslip. Two pathologists blinded to the clinical data reviewed the immunoreactivity for Ki-67 protein under a light microscope. The Ki-67 index was scored according to the percentage of stained tumor cells based on 2000-cell counts [Immunohistochemical staining for Ki-67 was performed using formalin-fixed, paraffin-embedded tumor tissue sections (3 μm thick) from 147 g-NENs Figure . Brieflyl counts  and was The patients were monitored after surgery by telephone calls, outpatient visits and letters. The overall survival (OS) time was calculated as the number of months from the date of surgery to the date of last contact, date of death for any cause, or date of end point. The recurrence free survival (RFS) time was calculated as the number of months from the date of surgery to the date of identification of disease recurrence , the date of death or last contact, or the date of end point. Our department follows a standardized surveillance protocol and follows up with patients at three-month intervals for the first two years, six-month intervals for years two to five, and at least once per year five years after surgery. The postoperative follow-up data included clinical symptoms and signs, laboratory tests, imagological examinations, and endoscopy and biopsy results. In this study, the median follow-up was 40 (range 2–106) months. The median survival time was 25 (range 2–106) months. The median RFS time was 21 (range 1–106) months.http://www.r-project.org) with the survival and rms package. A final model was selected using a backward step-down process, which used the Akaike information criterion as a stopping rule. The model performance for predicting outcome was evaluated by calculating the concordance index (C-index). Calibration of the nomogram for 1-, 3-, and 5-year RFS and OS was performed by comparing the predicted survival with the observed survival after bias correction. Bootstraps with 1,000 resample were used for these activities.On the basis of the results of the multivariable analysis, a nomogram was formulated by R 3.2.0 . Chi-square, Fisher's exact or unpaired Student's t tests were used to compare the difference between blood NLR and the clinicopathologic factors, as appropriate. A Pearson correlation was utilized to evaluate the relationship among blood NLR, Ki-67 index and recurrence. Univariate survival analysis was performed using the Kaplan-Meier method, and a log-rank test was used to assess the significance between groups. A multivariate survival analysis was performed using the Cox proportional hazards model. The significant variables from the univariate analysis were included in the model.